Cloning of a beta integrin subunit cDNA from an embryonic cell line derived from the freshwater mollusc, Biomphalaria glabrata.

A cDNA encoding an integrin subunit was cloned and structurally characterized from an embryonic cell line derived from Biomphalaria glabrata, snail intermediate host of the human blood fluke Schistosoma mansoni. Cells of the B. glabrata embryonic (Bge) snail cell line were initially tested for their sensitivity to the integrin-specific tetrapeptide inhibitor Arg-Gly-Asp-Ser (RGDS). Washed Bge cells when exposed to 0.5 to 2.0mM of RGDS were significantly inhibited in their ability to spread on a glass substrate. Spreading inhibition was specific, since a control peptide Arg-Gly-Glu-Ser (RGES) did not have the same effect. RT-PCR was performed using previously reported degenerate oligonucleotide primers to the ligand binding domain (LBD) of known beta integrin subunits and Bge cDNA. A 137 bp fragment was amplified, TA-cloned, sequenced, and the na and deduced aa sequences were compared with other beta integrins. Databank analysis showed that the 137 bp product shared >/=55.6% aa similarity to other beta integrin LBDs. Southern and northern blot analyses using the 137 bp sequence as a probe revealed binding to Bge genomic DNA restriction fragments and to an approximately 8 kb poly-(A)+RNA transcript, respectively. An exact 5' primer synthesized to the 137 bp product and an oligo-d(T) primer then were used to amplify from Bge cDNA, a partial beta integrin sequence of 2285 bp that contained a 1971 bp ORF. The remaining upstream coding region was obtained using 5' RACE methods. The complete ORF, consisting of 2364 bp, encoded a 788 aa sequence with shared similarity to other known beta integrins (44.6-61.5%). Sequence and structural comparisons, which include a characteristic LBD, a series of three homologous cysteine-rich repeats, membrane proximal sequence (LLTFIHD), cytoplasmic NPXY motifs, and predicted domain lengths of the molluscan protein, clearly identifies it as an integrin homologue. This report represents the first cloning of a cDNA putatively encoding an integrin subunit from molluscan cells, and establishes the Bge cell line as a model for studying cellular adhesion in molluscs at the molecular level.